Project Summary/Abstract Miami has emerged as the US epicenter of the now-global Zika virus (ZIKV) epidemic. The consequences of infection remain to be fully elucidated, but the probable link between ZIKV infection and fetal developmental complications raises enormous concern as to the potential impact of ZIKV. Current emergency use authorization (EUA) commercial assays detect ZIKV RNA, the presence of ZIKV-specific IgM, and utilize the laborious Plaque Reduction Neutralization Test (PRNT) to detect previous exposure to ZIKV. PRNT assay results took as long as 10 weeks to report back to the patient during the 2016 outbreak in Miami and can be equivocal for ZIKV exposure when patients have had a history of dengue virus (DENV). DENV is of considerable concern, because it infects approximately 200 million people a year, is spread by the same mosquito vector as ZIKV, and causes symptoms similar to ZIKV. This serological cross-reactivity between ZIKV and DENV is currently causing significant problems in the ZIKV diagnostic field. Accordingly, our plan is to develop a highly-specific diagnostic test for the detection of previous exposure to ZIKV. Fortunately, we are in a unique position to rapidly develop this diagnostic. We have isolated anti-ZIKV monoclonal antibodies (mAbs) from human volunteers that only recognize ZIKV and none of the four DENV serotypes. We have used one of these mAbs in a plasma competition assay to determine previous exposure to ZIKV. We have developed a rapid and innovative assay to detect previous flavivirus exposure that can discriminate between ZIKV and DENV with extraordinary specificity. In the current Phase I proposal, we will 1) advance our preliminary research results to create a clinical ZIKV diagnostic assay for serological reactivity in the convalescent phase, 2) test our innovative diagnostic assay on well-defined, complex human clinical samples, and 3) develop a novel ZIKV diagnostic test to distinguish between recent and older ZIKV infections.